Separation of DNA has been central to advances in our understanding of human biology and health. Genome sequencing, genotyping, phenotyping as well as metagenomic analysis of pathogenic organisms and microbial communities have all relied on methods for DNA separation, most commonly based on DNA fragment length. Same-length fragments must then be sequenced or separated by sequence. The goal of this proposal is to invent a microfluidic chip for simple, rapid, low-cost separation of DNA in two dimensions: length and sequence. This will be achieved by first separating DNA by length using conventional sieving polymers and then separating the same-length DNA fragments in the second dimension of sequence using a new gel medium that is formed by self-association of guanosine compounds. This guanosine gel, or G-gel, shows promise for separation of DNA on the basis of sequence without the need for mobility shifts induced by three-dimensional structure or resistance to thermal/chemical denaturation that are the basis for existing techniques. The specific aims of this proposal are to: (1) Use capillary gel electrophoresis to systematically investigate the DNA sequence selectivity of the G-gels and evaluate limits of sequence-based resolution; (2) Design and fabricate a microfluidic chip for the two-dimensional (2-D) DNA separation; (3) Individually optimize separations in each of the two dimensions; (4) optimize the combined, 2-D separation and evaluate limits of resolution; (5) test the ability of the chip to separate microbial DNA samples that have been fully characterized by standard methods. The ultimate goal is a self-contained, integrated device that would incorporate the 2-D separation with on-board power supply and detection into an expanded chip that would include pre-analysis steps for sample processing, including DNA extraction, purification and PCR. Realization of such an integrated platform would provide a true lab-on-a-chip for real-time DNA analysis that would offer important advantages for wide-ranging applications, for example, routine genomic analysis at point-of-care and in challenging settings such as remote areas or disaster sites, and for profiling pathogenic organisms and health-related microbial communities and monitoring their responses to environmental stressors.